High expression of serine protease, Brachyurin in the posterior midgut of black soldier fly (Hermetia illucens) during horse dropping processing

Objective Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. Results First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-024-06846-0.


Livestock droppings
We used three kinds of livestock droppings; horse, dairy cow, and laying hen.All of these droppings were collected from each livestock farm on the Fuchu campus.The horse was raised on timothy hay (Phragmites australis), alfalfa hay, rock salt, carrot, apple, and horse feed (Havens co. ltd., Vierlingsbeek, Netherlands).
Adult commercial laying hens (White Leghorn) aged 328 to 354 days were used.The hens were housed in individual cages measuring 25 cm × 40 cm × 43 cm (width × depth × height).
Room temperature was maintained at 25.0°C.Lighting was provided by fluorescent bulbs set at an intensity of 500 lux at the feeder.The light cycle was 14 hours light and 10 hours dark, with the lights on from 06:00 to 20:00.Hens had ad libitum access to water and feed.Commercial diets for laying hens were used.The droppings were collected and weighed.These hen droppings were then processed for compositional analysis and used as BSF larval diets.

Feeding tests
Newly hatched larvae were fed an artificial diet for ten-days.Then, 20 ten-day-old larvae were transferred to a new plastic cup.Ten grams of each livestock droppings was added to a plastic cup every three days until the larvae reached the prepupal stage.We recorded the body weight of the BSF larvae, and the food intake until the end of the experiment.The feed conversion ratio (FCR) was calculated as follows:  =  ℎ −  ℎ   ℎ   −   ℎ   Also, we performed feeding tests against newly hatched larvae (zero-day-old larvae).The feeding tests were carried out in triplicate as biological replicates.S3.Protease transcripts with value higher than 150.Ten grams of each livestock droppings was added to a plastic cup every three days until the larvae reached the prepupal stage.We recorded the body weight of the BSF larvae until the end of the experiment.The feeding tests were carried out in triplicates as biological replication.N.D mean not detect.All data are shown as the mean ± standard deviation (SD).The Tukey HSD method was used for determining statistical significance.
Table S5.The efficiency of processing livestock droppings using 10-day-old larvae.Newly hatched larvae were fed an artificial diet for ten-days.Then, 20 ten-days-old larvae were transferred to a new plastic cup.Ten grams of each livestock droppings was added to a plastic cup every three days until the larvae reached the prepupal stage.We recorded the body weight of the BSF larvae and the food intake until the end of the experiment.N.D mean not detect.All data are shown as the mean ± standard deviation (SD).The Tukey HSD method was used for determining statistical significance.

Fig. S1 A
Fig. S1 A pipeline for trypsin selection in H. illucens larvae.We constructed digestive enzymes extract pipeline using a public database, then extracted global proteases from the midgut transcriptome of H. illucens larvae.129,909 sequences were found, and then 470 sequences were extracted by HMM search using the trypsin motif.These transcripts were cut-off with TPM value below 150.Finally, 54 sequences were obtained and annotated using KEGG orthology and BLAST KOARA database.

Fig
Fig. S2 The midgut of the instar larva in H. illusens.The larval midgut was divided into three parts; anterior midgut (AMG), middle midgut (MMG), posterior midgut (PMG) according to the methods of Bonelli et al. (2019).

Table S4 .
The efficiency of processing livestock droppings using 0-day-old larvae.